Mu opioids, widely used for pain management and also abused, act through mu opioid receptors. Clinical differences in the actions of many these drugs raised the possibility of mu receptor subtypes, a concept confirmed by the identification of a vast array of both 3'and 5'splice variants from the mu opioid receptor (OPRM1) gene. The importance of 5'splicing has recently been demonstrated in an exon 11 knockout mouse. Loss of exon 11-associated variants had little effect upon morphine analgesia but diminished the activity of both heroin and morphine-62-glucuronide (M6G). Conversely, disruption of exon 1 led to complete loss of morphine action while heroin and M6G retained activity. 3'splicing, defined by splicing from exons 1/2/3 to alternative exons downstream of exon 3 (3'exons), generates 15 different carboxyl terminal variants in mouse. Similar C-terminal variants were identified in rat and human. The functional significance of these C-terminal variants has been suggested by differences in region-specific expressions at both mRNA and protein levels, pre- and post-synaptic localization and mu agonist-induced G protein coupling, phosphorylation and receptor internalization. However, the in vivo function of these C- terminal tails remains largely unknown. The primary goal of this proposal is to obtain a better understanding of the pharmacological function of carboxyl terminal tails generated by 3'alternative splicing in the OPRM1 gene through gene targeting in mice. Our objective is not to eliminate receptors, but only to disrupt the C-terminal tails produced by 3'splicing. Our targeting strategy is to insert stop codons into exons to generate truncated receptors without interfering with the 3'splicing. Our preliminary studies with minigene constructs indicated that these designed mutant stop codons are feasible to generate predicted mutant receptors lacking C-terminal tails in P19 and NIE-115 cells, promising success in targeted mice. The first aim will convert all the MOR-1 splice variants into identical truncated receptors by creating a stop codon at the end of exon 3. The second aim will limit truncation to variants containing C-terminal tails encoded by either exon 4 or exon 7 by creating the stop codon at the beginning of the respective exon. Although the approach could be used on all the variants, we choose to focus on exon 4 and exon 7 because these exons are the most highly conserved 3'exons and responsible for the vast majority of MOR-1 variants in the mouse brain. The third aim will characterize these mutant mice through establishing distribution and expression of the mutant receptors, and determining their opioid binding and G protein coupling profiles. By generating these models, we hope to provide useful tools to investigate the in vivo functions of the C-terminal tails. Understanding the in vivo function of the C-terminal tails will provide new insights into the structural and functional relationships of the mu opioid receptors, and into the complex actions of opioids in animals and humans, possibly leading to new targets for developing drugs used in control of pain and drug of abuse. PUBLIC HEALTH RELEVANCE: The primary goal of this proposal is to obtain a better understanding of the pharmacological function of carboxyl terminal tails generated by 3'alternative splicing in the OPRM1 gene through gene targeting in mice. The proposed targeted mouse models will provide useful tools to investigate the in vivo functions of the C-terminal tails. Understanding the in vivo function of the C-terminal tails will provide new insights into the structural and functional relationships of the mu opioid receptors, and into the complex actions of opioids in animals and humans, possibly leading to new targets for developing drugs used in control of pain and drug of abuse.